368 J. Agric. Food Chem. 1998, 46, 368−375 Survey of the Free and Conjugated Myricetin and Quercetin Content of Red Wines of Different Geographical Origins Morag S. McDonald,† Mark Hughes,† Jennifer Burns,† Michael E. J. Lean,§ David Matthews,‡ and Alan Crozier*,† Plant Molecular Science Group, Bower Building, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, U.K., Department of Human Nutrition, University of Glasgow, Queen Elizabeth Building, Royal Infirmary, Glasgow G31 2ER, U.K., and Safeway plc, Beers, Wines, Spirits and Tobacco Unit, 6 Millington Road, Hayes, Middlesex UB3 4AY, U.K. Gradient reversed-phase HPLC was used to obtain quantitative estimates of the levels of free and conjugated myricetin and quercetin in 65 red wines from Italy, Chile, France, California, Australia, Bulgaria, Spain, Romania, New Zealand, Brazil, Morocco, and Hungary. The concentrations of total flavonols ranged from 4.6 to 41.6 mg L-1. High total flavonol levels appear to be associated with the use of thick-skinned grape varieties, such as Cabernet Sauvignon, with a high skin:volume ratio, which were left to ripen fully in sunny conditions before harvest and which were extracted efficiently by modern methods of vinification. Some Chilean Cabernet Sauvignon wines contained up to 40 mg of total flavonols L-1, which was higher than the levels detected in Cabernet Sauvignon from France, California, and Australia. The flavonol content of 1989 and 1990 Cabernet Sauvignon from Bulgaria was <6mgL-1. Chilean Cabernet Sauvignon, Merlot, and Pinot Noir all contained consistently higher concentrations of flavonols than their counterparts from different geographical regions. Keywords: HPLC; quantitative analysis; flavonols; myricetin; quercetin; red wines INTRODUCTION sundara, 1992), and their consumption has been associ- ated with a reduced risk of cancer (Verma et al., 1988; Flavonoids are secondary metabolites found ubiqui- Wattenburg, 1985, 1990; Wei et al., 1990), thrombosis tously in plants (Harborne, 1994), where they function (Gryglewski et al., 1987; Laughton et al., 1991), and as UV-B protectants [see Koes et al. (1994)] and are cardiovascular disease (Gregory et al., 1990; Hertog, involved in the regulation of pollen tube growth in the stigma (Mo et al., 1992; Vogt et al., 1994). They also 1994). Fruits, vegetables, and beverages are important act as regulatory signals in the transcription of nodu- dietary sources of flavonoids, especially flavonols (Her- lation genes in Rhizobium cells as a first step toward tog et al., 1992a, 1993b; Crozier et al., 1997b) with - legume root nodule formation and symbiotic nitrogen beverages accounting for at least 25 30% of the total fixation (Firmin et al., 1986). The metal-chelating daily intake (Hertog et al., 1993b). Recent epidemio- capability of flavonoids, together with their free-radical- logical studies have suggested that flavonol-rich diets scavenging properties, has led to proposals that fla- are associated with higher life expectancy (Hertog et al., vonoids act as dietary antioxidants (Bors and Saran, 1993a, 1995; Hertog and Hollman, 1996). 1987). It is this role, as potential inhibitors of free- Red wines are a rich source of flavonoids (Revilla et radical-mediated diseases, such as coronary heart dis- al., 1986; Hertog et al., 1993b; Frankel et al., 1996), and ease, stroke, and cancers, and the associated health studies have shown that moderate consumption is benefits of a high-flavonoid diet, which has received the associated with a reduced risk of coronary heart disease most attention in recent literature. (Stampfer et al., 1988; Gronbaek et al., 1995). In many Flavonoids are phenolic compounds which occur pri- countries a high intake of saturated fats has been linked marily as glycosides (Markham, 1989). They are clas- to elevated levels of coronary heart disease, yet this sified into two major groups: the anthocyanins and the relationship does not apply to certain regions of France, anthoxanthins. The anthoxanthins are subdivided into where red wine is consumed in preference to beer, a the flavones, flavanones, and flavonols (Herrmann, phenomenon popularly referred to as the “French 1988). Flavonols, such as quercetin (I; Chart 1), myrice- paradox” (Renaud and Lorgeril, 1992; Renaud, 1996). tin (II), isorhamnetin (III), and kaempferol (IV), and Maxwell et al. (1994) reported a rapid increase in the the corresponding flavones, apigenin (V), and luteolin (VI), have antioxidant properties (Shahidi and Wana- antioxidant activity in the serum of 10 human volun- teers after drinking red Bordeaux wine. Further studies on this observation have indicated that it is the phenolic * Author to whom correspondence should be addressed components in red wine, rather than the alcohol content, [telephone, (-44) 141-330-4613; fax, (-44) 141-330-4447; e-mail, [email protected]]. which exert the protective effects (Sato et al., 1996). The † University of Glasgow. phenolics in red wines inhibit the oxidation and cyto- § Royal Infirmary. toxicity of low-density lipoproteins in vitro which may ‡ Safeway plc. decrease their artheoreogenicity and explain the re- S0021-8561(97)00677-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/22/1998 Free and Conjugated Flavonol Content of Red Wines J. Agric. Food Chem., Vol. 46, No. 2, 1998 369 Chart 1. Structures of Quercetin, Myricetin, Isorhamnetin, Kaempferol, Apigenin, Luteolin, trans-Resveratrol, Gallic Acid, (+)-Catechin, (-)-Epicatechin, and Morin duced risk of coronary heart disease (Frankel et al., in wines has been described by Hertog et al. (1992b) following 1993a, 1995). an earlier detailed study by Harborne (1965) on the release of One non-flavonoid phenolic that is found in red wine, free flavonoids by acid and enzymic hydrolyses. In the present the stilbene resveratrol (VII), has been the focus of investigation, preliminary screenings were carried out to interest in the popular press (Jones, 1995; McWhirter, ascertain the most effective acid hydrolysis conditions. As a 1996) because of its ability to prevent platelet aggrega- result, all samples were hydrolyzed at 90 °C for2hin1.2M tion in coronary arteries (Kimura et al., 1985) and lessen HCl in 50% aqueous methanol, containing morin (XI)asan internal standard and 20 mM sodium diethyldithiocarbamate the incidence of cancer (Jang et al., 1997). However, as an antioxidant. A microscale hydrolysis procedure was used the hydroxybenzoate gallic acid (VIII) and certain + which involved adding a 300-µL sample of wine to a 3-mL glass flavonoids, most notably the flavan-3-ols ( )-catechin V-vial together with 400 µL of 6 M HCl, 300 µL of distilled (IX) and (-)-epicatechin (X), as well as the flavonols water, and 1 mL of methanol containing 40 mM sodium quercetin and myricetin, are present in red wines in far diethyldithiocarbamate and 5 µg of morin. A Teflon-coated higher concentrations (Frankel et al., 1995; Frankel, magnetic stirrer was placed in the vial which was sealed 1996; Lamuela-Ravento´s et al., 1995; Goldberg et al., tightly with a PTFE-faced septum prior to heating in a Reacti- 1995) and exhibit similar, if not greater, antioxidant and Therm heating/stirring module (Pierce, Rockford, IL). Extract antiplatelet aggregation activity than resveratrol [see aliquots of 100 µL, taken both before and after acid hydrolysis, Frankel et al. (1993a,b, 1995), Vinson et al. (1995), were made up to 250 µL with distilled water adjusted to pH Teissedre et al. (1996)]. 2.5 with trifluoroacetic acid and filtered through a 0.2-µm The present study, which utilized improved high- Anopore filter (Whatman, Maidstone, Kent, U.K.), prior to the 1 performance liquid chromatography (HPLC) procedures analysis of 100-µL volumes ( /50 aliquot of total sample) by (Crozier et al., 1997a) to investigate the flavonol content gradient elution reversed-phase HPLC. of 65 red wines, of wide-ranging geographical origin, High-Performance Liquid Chromatography. Samples demonstrates that the wines exhibit marked and sys- were analyzed using a Shimadzu (Kyoto, Japan) LC-10A series temic differences in the levels of both free and conju- automated liquid chromatograph comprising a SCL-10A sys- gated myricetin and quercetin. tem controller, two LC-10A pumps, a SIL-10A autoinjector with sample cooler, a CTO-10A column oven, and a SPD-10A UV-vis detector linked to a Reeve Analytical (Glasgow, U.K.) METHODS 2700 data handling system. Reversed-phase separations were × Wines. Sixty-five bottles of red wine were purchased from carried out at 40 °C using a 150- 3.0-mm i.d., 4-µm Genesis × Allders Duty Free Shop, Glasgow Airport, Abbottsinch, Pais- C18 cartridge column fitted with a 10- 4.0-mm i.d., 4-µm ley, U.K.; Safeway plc, 373 Byers Rd., Glasgow, U.K.; Oddbins, C18 Genesis guard cartridge in an integrated holder (Jones 181 Byers Rd., Glasgow G12, U.K; J. Sainsbury’s plc, 10 Chromatography, Mid-Glamorgan, U.K.). The mobile phase Darnley Mains Rd., Glasgow G41, U.K.; Peckham & Rye, 21 was a 20-min, 20-40% gradient of acetonitrile in water Clarence Dr., Glasgow G12, U.K.; Victoria Wine Cellars, 159 adjusted to pH 2.5 with trifluoroacetic acid, eluted at a flow Hyndland Rd., Glasgow, U.K.; Tesco Stores Ltd., Telegraph rate of 0.5 mL min-1. Column eluent was monitored at 365 Rd., Heswall, The Wirral, Cheshire, U.K. nm which provided a limit of detection of <5 ng (Crozier et Extraction and Hydrolysis Conditions. Optimization al., 1997a). Linear 5-250-ng calibration curves were obtained of acidic conditions for the hydrolysis of flavonol conjugates for the flavonol aglycons morin, myricetin, quercetin, kaempfer- 370 J. Agric. Food Chem., Vol. 46, No. 2, 1998 McDonald et al. Table 1. Details of Red Wines Analyzed for Free and Conjugated Flavonol Contenta wines year price (£) label, region/country of origin; grapes; supplier 1, Valpolicella 1994 3.89 Gaetane Carron, Veneto, Italy; Corvina (70% max) with Rondinella and Molinara; Safeway 2, Valpolicella 1994 3.99 Fratelli Pasqua SpA, Verona, Italy; Corvina (70% max) with Rondinella and Molinara; Victoria Wine Cellars 3, Barolo 1993 7.99 Barolo, Cantina Terre del Barolo, Piedmont, S.